Means and methods for producing high affinity antibodies

ABSTRACT

Described are means and methods for producing high-affinity antibodies against an antigen of interest, using stable B-cell cultures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/EP2011/071676, filed Dec. 2, 2011,designating the United States of America and published in English asInternational Patent Publication WO 2012/072814 A1 on Jun. 7, 2012,which claims the benefit under Article 8 of the Patent CooperationTreaty to European Patent Application Serial No. 10193562.5, filed Dec.2, 2010 and under Article 8 of the PCT and under 35 U.S.C. §119(e) toU.S. Provisional Patent Application Ser. No. 61/419,909, filed Dec. 6,2010.

TECHNICAL FIELD

The disclosure relates to the field of cell biology. More specifically,it relates to the field of antibody production.

BACKGROUND

Ex vivo B-cell cultures are important tools in current biological andmedical applications. One important application is culturingantibody-producing cells in order to harvest antibodies, preferablymonoclonal antibodies. Monoclonal antibodies (mAbs) represent multipleidentical copies of a single antibody molecule. Amongst the benefits ofmAbs is their specificity for the same epitope on an antigen. Thisspecificity confers certain clinical advantages on mAbs over moreconventional treatments while offering patients an effective,well-tolerated therapy option with generally low side effects. Moreover,mAbs are useful for biological and medical research.

Mature B-cells can be cultured in vitro under conditions that mimic somekey aspects of the germinal center (GC) reaction; that is, activation ofB-cells with CD40 ligand (L) and the presence of cytokines likeinterleukin (IL)-4, IL-10 or IL-21. While B-cells cultured with CD40L,IL-2 and IL-4 produce very little Ig, addition of IL-21 leads todifferentiation to plasma cells accompanied by high Ig secretion.Although this in vitro system has proven useful to study some aspects ofB-cell differentiation, both naïve IgD+ B-cells and switched IgD-memoryB-cells eventually differentiate into terminally differentiated plasmacells, which is accompanied by cell cycle arrest precluding thegeneration of long-term antigen-specific BCR-positive cell lines.

Recent advances have provided insight into how multiple transcriptionfactors, including B-lymphocyte-induced maturation protein 1 (BLIMP1)and B-cell lymphoma (BCL)6 control development of GC B-cells intoterminally arrested, antibody-producing plasma cells. Thetranscriptional repressor BCL6 has been shown to prevent plasma celldifferentiation. BCL6 is highly expressed in GC B-cells where itfacilitates expansion of B-cells by down-regulating p53 and preventspremature differentiation of GC cells into plasma cells by negativelyregulating BLIMP1.

An improved method for generating an antibody-producing plasmablast-likeB-cell was recently described in WO 2007/067046, which is herebyincorporated by reference. According to this method, the amount of BCL6and a Bcl-2 family member, preferably Bcl-xL, are modulated in a B-cell,preferably a memory B-cell, to generate an antibody-producingplasmablast-like B-cell. In WO 2007/067046, the amount of BCL6 and/orBcl-xL expression product is either directly or indirectly influenced.Preferably, the amounts of both BCL6 and Bcl-xL expression productswithin the antibody-producing cell are increased, since both expressionproducts are involved in the stability of an antibody-producing B-cell.Bcl-xL is a member of the anti-apoptotic Bcl-2 family. Processes thatare controlled by the Bcl-2 family, which includes both pro- andanti-apoptotic proteins, relate to the mitochondrial pathway ofapoptosis. This pathway proceeds when molecules sequestered between theouter and inner mitochondrial membranes are released into the cytosol bymitochondrial outer membrane permeabilization. The pro-apoptotic familymembers can be divided in two classes. The effector molecules Bax andBak, which contain so-called Bcl-2 homology domain 3 (BH3) domains, areinvolved in permeablilizing the outer mitochondrial membrane by formingproteolipid pores; the pro-apoptotic BH3-only proteins (Bad, Bik, Bim,Bid, Hrk, Bmf, bNIP3, Puma and Noxa) function upon different cellularstresses by protein-protein interactions with other (anti-apoptotic)Bcl-2 family members.

Anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, Bcl-w, A1 and Mcl-1are generally integrated with the outer mitochondrial membrane. Theydirectly bind and inhibit the pro-apoptotic Bcl-2 proteins to protectmitochondrial membrane integrity.

In such a method, it is further preferred that the antibody-producingplasmablast-like B-cell is incubated with IL 21 and CD40L. A B-cell,such as an antibody-producing plasmablast-like B-cell, is preferablycultured in the presence of CD40L since replication of most B-cells isfavored by CD40L. It is furthermore preferred that STAT3 is activated inthe antibody-producing B-cell. Activation of STAT3 can be achieved in avariety of ways. Preferably, STAT3 is activated by providing anantibody-producing cell with a cytokine. Cytokines, being naturallyinvolved in B-cell differentiation, are very effective in regulatingSTAT proteins. Very effective activators of STAT3 are IL-2, IL-10, IL-21and IL-6, but also IL-7, IL-9, IL-15, IL-23 and IL-27 are known toactivate STAT3. Additionally, or alternatively, STAT3 activation isaccomplished by transfer into a B-cell of a nucleic acid encoding amutant of STAT3 that confers constitutive activation to STAT3. (Sean A.Diehl, Heike Schmidlin, Maho Nagasawa, Simon D. van Haren, Mark J.Kwakkenbos, Etsuko Yasuda, Tim Beaumont, Ferenc A. Scheeren, HergenSpits STAT3-mediated up-regulation of BLIMP1 is coordinated with BCL6down-regulation to control human plasma cell differentiation. J.Immunol. 2008 vol. 180 (7) pp. 4805-15.)

Most preferably, IL-21 is used, since IL-21 is particularly suitable forinfluencing the stability of an antibody-producing plasmablast-likeB-cell. In addition to up-regulating STAT3, IL-21 is capable ofup-regulating BLIMP1 expression even when BLIMP1 expression iscounteracted by BCL6. With the methods disclosed in WO 2007/067046, ithas become possible to increase the replicative life span of anantibody-producing cell since it is possible to maintain a B-cell in adevelopmental stage wherein replication occurs. In earlier ex vivoB-cell cultures, the replicative life span was only a few weeks to twomonths. During this time, the cultured cells lose their capability ofreplicating and die. With a method as disclosed in WO 2007/067046,however, it has become possible to prolong the replicative life span ofantibody-producing memory B-cells, so that ex vivo cultures aregenerated comprising plasmablast-like B-cells that are capable ofreplicating and producing antibody.

Although these methods enable the production of antibodies thatefficiently target an antigen of interest, improvement of antibodycharacteristics, such as binding affinity, is often desired. Bindingcharacteristics are, therefore, regularly altered by introducingmutations in the encoding nucleic acid, preferably in the CDR encodingregion, and testing the resulting antibodies. This is, however, timeconsuming. Alternative methods for obtaining high-affinity antibodiesare, therefore, desired.

DISCLOSURE

Provided are methods for producing and/or selecting high-affinityantibodies.

Provided are means and method for obtaining a B-cell population,starting from a given B-cell culture, which population has a higheraverage binding capacity than the original B-cell culture. Preferably, amonoclonal B-cell population is produced, starting from a monoclonalB-cell culture. Provided is a simple and elegant way of obtaining B-cellpopulations with an increased average binding capacity, without the needfor laborious mutation techniques.

Provided is a method for producing antibodies specific for an antigen ofinterest comprising:

-   -   a) selecting a B-cell capable of producing antibody specific for        the antigen of interest or selecting a B-cell capable of        differentiating into a B-cell that is capable of producing        antibody specific for the antigen of interest;    -   b) inducing, enhancing and/or maintaining expression of BCL6 in        the B-cell;    -   c) inducing, enhancing and/or maintaining expression of an        anti-apoptotic nucleic acid in the B-cell;    -   d) allowing expansion of the B-cell into a population of the        B-cells;    -   e) selecting at least one B-cell from the population of B-cells        producing a B-cell receptor and/or antibody with a binding        capacity higher than the average binding capacity of the        population of B-cells for the antigen of interest;    -   f) culturing the at least one B-cell into a population of        B-cells; and    -   g) obtaining antibodies produced by the B-cell culture.

Within a population of monoclonal B-cells capable of producing antibodyspecific for an antigen of interest, it is possible to select, in stepe) of a method hereof, at least one, optionally more than one, such as,for instance, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25 or 50 B-cells with abinding capacity for the antigen of interest that is higher than theaverage binding capacity of the population of B-cells for the antigen ofinterest. Such B-cells with a higher binding capacity for an antigen ofinterest than the average binding capacity of the population of B-cellsfor the antigen of interest are herein also called “high-affinityB-cells.” One possible reason for a difference in binding capacitybetween multiple B-cells in a monoclonal population of B-cells is thatthe expression of the BCR varies between B-cells in the population. AB-cell with a relatively high expression of the BCR will bind moreantigen of interest than a B-cell with a relatively low expression ofthe BCR. However, it is expected that antibodies produced by B-cellswith different expression of the BCR have the same binding affinity. Thepresent inventors surprisingly found that, besides a relatively high BCRexpression, a collection of high-affinity B-cells produce antibodiesspecific for the antigen of interest that bind the antigen with a higheraffinity than the average affinity of antibodies produced by thepopulation of B-cells. Even more surprisingly, the inventors found thatthe B-cell cultures obtained with a method hereof contained cells thatbound antigen with a higher affinity than the average B-cell in theoriginal culture. Single B-cells can thus be isolated from a givenB-cell population on the basis of their higher binding capacity bymethods known in the art and be expanded to a new B-cell population inat least three weeks. These new B-cells produce antibodies that have ahigher affinity than the antibodies produced by the original B-cellpopulation that the new B-cells are derived from. This finding iscontrary to expectations because a person skilled in the art wouldexpect that after isolation of one B-cell (subclone) from an alreadymonoclonal population of B-cells, the affinity for the antigen ofantibody produced by the progeny of the subclone of the alreadymonoclonal B-cell population will return to the average affinity for theantigen, comparable to the average affinity of the population of B-cellsfrom which the at least one B-cell was selected.

Thus, in one embodiment in step a) of a method hereof, preferably asingle B-cell is selected, for instance, from a polyclonal population ofB-cells. The single B-cell is subsequently expanded into a monoclonalpopulation of B-cells in steps b) to d). This is, for instance, achievedusing a method as described in WO 2007/067046, which is discussedhereinbefore. Hence, in step d), a monoclonal B-cell line specific foran antigen of interest is obtained. In principle, all B-cells in themonoclonal B-cell line produce essentially the same antibodies specificfor the antigen, although small differences in the affinity for theantigen may be present between cells of the monoclonal B-cell line,i.e., some B-cells in the monoclonal population produce antibodies withan affinity that is slightly higher than the average affinity and someB-cells in the monoclonal population produce antibodies with a slightlylower affinity. The population of B-cells becomes slightly heterogeneousagain. In step e), at least one of such B-cells with a higher affinitythan the average affinity is selected from the monoclonal B-cell line.In step f), the B-cell or B-cells selected in step e) are subsequentlycultured into a second, preferably monoclonal, B-cell line. Provided isthe insight that this second, preferably monoclonal, B-cell line has anaverage affinity that is higher than the average affinity of theoriginal monoclonal B-cell population obtained in step d). As describedabove, it was surprisingly found that the high affinity of a selectedB-cell is maintained after culturing, even if culturing takes placeduring a prolonged period of time, instead of returning to the averageaffinity of the original population. Thus, the second monoclonalpopulation of B-cells cultured in step f) has a higher average affinityfor the antigen than the monoclonal population of B-cells cultured instep d). Similarly, the affinity of most B-cells in the secondmonoclonal population of step f) is higher than the affinity of mostB-cells in a monoclonal population of step d).

Thus provided in one embodiment is a method for obtaining a B-cellpopulation with an increased average affinity for an antigen ofinterest, as compared to an original monoclonal B-cell population with agiven average affinity for the antigen of interest, the methodcomprising:

-   -   providing a monoclonal B-cell population that is specific for        the antigen of interest,    -   selecting at least one B-cell from the population of B-cells        producing a B-cell receptor and/or antibody with a binding        capacity higher than the average binding capacity of the        population of B-cells for the antigen of interest; and    -   culturing the at least one B-cell into a population of B-cells.

Further provided is a method for producing antibodies specific for anantigen of interest, the method comprising:

-   -   a) selecting a single B-cell capable of producing antibody        specific for the antigen of interest or selecting a B-cell        capable of differentiating into a B-cell that is capable of        producing antibody specific for the antigen of interest;    -   b) inducing, enhancing and/or maintaining expression of BCL6 in        the B-cell;    -   c) inducing, enhancing and/or maintaining expression of an        anti-apoptotic nucleic acid in the B-cell;    -   d) allowing expansion of the B-cell into a first monoclonal        B-cell line;    -   e) selecting from the first monoclonal B-cell line at least one        B-cell that produces a B-cell receptor and/or antibody with a        binding capacity for the antigen of interest higher than the        average binding capacity of the first monoclonal B-cell line;    -   f) culturing the at least one B-cell selected in step e) into a        second, preferably monoclonal, B-cell line; and    -   g) obtaining antibodies produced by the second, preferably        monoclonal, B-cell line.

Antibodies are obtained that have an affinity for the antigen ofinterest that is higher than the average affinity for the antigen ofinterest of antibodies produced by B-cells of the first monoclonalB-cell line.

In another embodiment, more than one B-cell is selected in step a) of amethod hereof; for instance, 2, 3, 4, 5, 10, 15, 25, 50 or 100 B-cells.The B-cells are, for instance, selected from a polyclonal population ofB-cells or from a biological sample. The selected B-cells aresubsequently expanded into a population of B-cells in steps b) to d),for instance, using a method as described in WO 2007/067046. Theobtained B-cell population is thus a (second) polyclonal B-cellpopulation. Thereafter, and before step e) of a method hereof is carriedout, a monoclonal population of B-cells is preferably produced. This is,for instance, done by selecting a single B-cell from the (second)polyclonal population of B-cells using Fluorescence Activated CellSorting or limiting dilution, which are explained hereinbelow, andexpanding the selected single B-cell to a monoclonal population ofB-cells. Then, step e) of a method hereof is carried out, in which atleast one B-cell with a higher affinity than the average affinity of themonoclonal B-cell population is selected. In step f), the B-cell orB-cells selected in step e) are subsequently cultured into a secondmonoclonal B-cell line, after which, antibodies produced by the secondmonoclonal B-cell line are obtained in step g).

A method as described herein allows for obtaining improved,high-affinity antibodies, preferably monoclonal antibodies, without theuse of recombinant techniques. Before the instant disclosure, affinityof (monoclonal) antibodies is increased using such recombinanttechniques. The sequence of the nucleic acid encoding the antibody firstneeds to be determined. Subsequently, one or more mutations areintroduced into the sequence of the nucleic acid encoding the antibody.Then, the genes containing one or more mutations need to be expressed ina cell followed by production of antibodies in producer cells. Finally,the mutated antibody has to be tested for its binding capacity to theantigen of interest in order to determine whether antibody with animproved affinity for the antigen as compared with the non-mutatedantibody is obtained. Such a process for improving the affinity of anantibody is elaborate and time consuming. A method according to theinstant disclosure allows the production of high-affinity antibody in astraight-forward and less elaborate process without the need ofmolecular engineering.

In one embodiment hereof, after the step of selecting at least onehigh-affinity B-cell from the already monoclonal population of B-cells(step e) of a method hereof as described above), the at least onehigh-affinity B-cell is allowed to expand into a population of B-cells,preferably a monoclonal B-cell line, again, after which another step ofselecting at least one high-affinity B-cell from the new population ofB-cells, preferably from the new monoclonal B-cell line, is performed.By repeating the steps of allowing expansion of a selected B-cell into apopulation and selecting at least one B-cell on the basis of its bindingcapacity for an antigen, i.e., repeating steps d) and e), it is possibleto generate high-affinity antibody-producing B-cells. Preferably, byrepeating the steps of expansion and selection as described above, it ispossible to increase with each selection cycle the affinity of antibodyproduced by the resulting B-cell population for the antigen of interest.

A method is thus provided comprising, following step e) of a methodhereof, repeating the step of allowing expansion of at least oneselected high-affinity B-cell into a population of B-cells, preferably amonoclonal B-cell line, and selecting again at least one high-affinityB-cell, i.e., repeating steps d) and e) of a method hereof at leastonce. The steps are, for instance, repeated once, but preferably twice,three times, four times, five times or even more times.

In one embodiment, a method hereof is provided wherein the at least oneB-cell selected in step e) is cultured for at least four weeks.Preferably, the at least one B-cell selected in step e) is cultured forat least six weeks, more preferably for at least nine weeks, morepreferably for at least three months, more preferably for at least sixmonths.

Without being bound to any theory, it is believed that differences inthe affinity of antibodies for an antigen of interest within apopulation of monoclonal B-cells may result from processes mediated byActivation Induced Cytidine Deaminase (AID). Antigen-activated naïve andmemory B-cells in the germinal center undergo extensive proliferation,accompanied by somatic hypermutations (SHM) and class-switchrecombination (CSR) of Ig genes mediated by AID. AID deaminatesdeoxycytidine residues in immunoglobulin genes, which triggers antibodydiversification. It was demonstrated in patent application US2008305076that IL 21 induces BLIMP, BCL6 and AID expression, but does not directlyinduce somatic hypermutation. However, the present inventors found thatAID is expressed in B-cells that are cultured according to a method asherein described. The expression of AID in (a B-cell that will developinto) an antibody-producing B-cell allows the generation of novelimmunoglobulins that harbor mutations that were not present in theoriginal B-cell before transduction with BCL6 and an anti-apoptoticnucleic acid. Thus, culturing B-cells in which somatic hyper mutation isinduced by expression of AID allows the generation of immunoglobulinvariants that, for example, have a higher or lower affinity for anantigen of interest, or that are more stable, for example, in an aqueoussolution or under increased salt conditions, or any combination thereof.

Upon selection of at least one high-affinity B-cell from the populationof B-cells, AID is still expressed within the selected at least oneB-cell. Therefore, after selection of such a B-cell, AID in the B-cellstill allows the introduction of mutations in the immunoglobulin gene ofthe progeny of the B-cell. Somatic hypermutations in immunoglobulingenes occur preferentially in the CDR3 region of the Ig genes. Mutationsintroduced in the CDR3 region of the immunoglobulin are more likely toresult in a reduced or lost binding affinity for an antigen of theimmunoglobulin than in an increased binding affinity. The presentinventors, however, did find increased binding affinity.

As used herein, the term “anti-apoptotic nucleic acid” refers to anucleic acid that is capable of delaying and/or preventing apoptosis ina B-cell. Preferably, the anti-apoptotic nucleic acid is capable ofdelaying and/or preventing apoptosis in an antibody-producing B-cell.Preferably, an anti-apoptotic nucleic acid is used that comprises anexogenous nucleic acid. This means that either a nucleic acid sequenceis used that is not naturally expressed in B-cells, or that anadditional copy of a naturally occurring nucleic acid is used, so thatexpression in the resulting B-cells is enhanced as compared to naturalB-cells. Various anti-apoptotic nucleic acids are known in the art, sothat various embodiments are available. Preferably, a gene encoding ananti-apoptotic molecule is used. More preferably, a nucleic acid is usedthat is an anti-apoptotic member of the Bcl-2 family becauseanti-apoptotic Bcl-2 proteins are good apoptosis inhibitors. Manyprocesses that are controlled by the Bcl-2 family (which family includesboth pro- and anti-apoptotic proteins) relate to the mitochondrialpathway of apoptosis, as outlined in more detail hereinbelow.Anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, Bcl-w, A1 and Mcl-1are preferred because they are generally integrated with the outermitochondrial membrane. They directly bind and inhibit the pro-apoptoticproteins that belong to the Bcl-2 family to protect mitochondrialmembrane integrity.

In a particularly preferred embodiment, the anti-apoptoticpolynucleotide encodes Bcl-xL and/or Mcl-1 and/or a functional part ofBcl-xL and/or a functional part of Mcl-1. A combination of BCL6 andBcl-xL nucleic acids, as well as a combination of BCL6 and Mcl-1 nucleicacids, is particularly suitable for immortalizing B-cells and long-termculture of the resulting plasmablast-like B-cells. Most preferably, theanti-apoptotic nucleic acid encodes Bcl-xL or a functional part thereof,because a combination of BCL6 and Bcl-xL stabilizes B-cells particularlywell.

A functional part of Bcl-xL and a functional part of Mcl-1 are definedherein as fragments of Bcl-xL and Mcl-1, respectively, that haveretained the same kind of anti-apoptotic characteristics as full-lengthBcl-xL and Mcl-1, respectively, in kind (but not necessarily in amount).Functional fragments of Bcl-xL and Mcl-1 are typically shorter fragmentsof Bcl-xL and Mcl-1, which are capable of delaying and/or preventingapoptosis in a B-cell. Such functional fragments are, for instance,devoid of sequences that do not contribute to the anti-apoptoticactivity of Bcl-xL or Mcl-1.

A population of B-cells hereof preferably is a monoclonal population ofB-cells. An example of a population of B-cells hereof is a cell line ofB-cells, preferably monoclonal B-cells. Hence, a population of B-cellshereof is most preferably a monoclonal B-cell line. Allowing expansionof the B-cell into a population of the B-cells is, for instance,accomplished by culturing the B-cell until a population of the B-cellsis obtained.

Within a population of B-cells, even in a population of monoclonalB-cells, the binding capacity of the BCRs of the B-cells of thepopulation, and the binding capacity of the antibodies produced by theB-cells of the population, is not equal. Instead, variation in thebinding capacity exists. The average binding capacity of a population ofB-cells is herein defined as the average of the binding capacity oraverage affinity of the BCR and/or antibody of all individual B-cells inthe population. The average affinity for an antigen of interest of anantibody produced by a B-cell or by a population of B-cells is hereindefined as the average of the affinities for the antigen of interest ofthe antibodies produced by all individual B-cells in the population. Ahigh-affinity B-cell from a population of B-cells hereof, preferablyfrom a monoclonal B-cell line, is preferably selected from the upper 40%of the B-cells of a population, preferably of a monoclonal B-cell line,with respect to binding capacity and/or affinity, preferably from theupper 30% of the B-cells of the population or monoclonal B-cell line,more preferably from the upper 25% of the B-cells of the population ormonoclonal B-cell line, more preferably from the upper 20% of theB-cells of the population or monoclonal B-cell line, more preferablyfrom the upper 15% of the B-cells of the population or monoclonal B-cellline, more preferably from the upper 10% of the B-cells of thepopulation or monoclonal B-cell line, more preferably from the upper 1%of the B-cells of the population or monoclonal B-cell line. In oneembodiment, one high-affinity B-cell is selected from the upper 1% ofthe B-cells of a population or monoclonal B-cell line with respect tobinding capacity and/or affinity.

The average affinity for an antigen of interest of antibody produced bya population of B-cells, preferably by a monoclonal B-cell line,cultured from at least one high-affinity B-cell hereof is preferably atleast 1.1 times the average affinity for the antigen of interest of thepopulation of B-cells from which the at least one high-affinity B-cellwas selected, more preferably at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 10.0, 20,50, 100 times, or more, the average affinity for the antigen ofinterest.

The affinity of an antibody can be determined using any method known toa person skilled in the art. The affinity of an antibody is, forinstance, determined using Enzyme-linked immunosorbent assay (ELISA),Surface Plasmon Resonance (such as Biacore) or Octet (ForteBio). SurfacePlasmon Resonance (SPR) and Octet are techniques to measure biomolecularinteractions in real-time in a label-free environment. For SPR, one ofthe interactants, for instance, an antibody, is immobilized to thesensor surface, the other, for instance, antigen, is free in solutionand passed over the surface. Association and dissociation is measured inarbitrary units and preferably displayed in a sensorgram. Any change inthe number of molecules bound to the biosensor tip causes a shift in theinterference pattern that can be measured in real-time. Using Octet, theinterference pattern of white light reflected from two surfaces, a layerof immobilized protein on the biosensor tip, and an internal referencelayer is analyzed. The binding between a ligand immobilized on thebiosensor tip surface, for instance, an antibody, and a protein insolution, for instance, an antigen of interest, produces an increase inoptical thickness at the biosensor tip, which results in a wavelengthshift that is a direct measure of the change in thickness of thebiological layer. ELISA comprises immobilizing a protein, for instance,the antigen of interest, on the surface of the solid support, forexample, a 96-well plate, and applying a sample to be detected orquantified on the solid support. Alternatively, a capture antibody isfixated on the surface of a solid support after which a samplecontaining the protein to be detected or quantified is applied to theimmobilized capture antibody allowing the protein of interest to bind.Non-binding proteins are then washed away. Subsequently, a specificantibody conjugated to a label or an enzyme (or a primary antibodyfollowed by a secondary antibody conjugated to a label or an enzyme) isadded to the solid support. Preferably, the affinity constant (K_(D)) ofan antibody produced by a B-cell hereof is determined.

Binding of a B-cell hereof to an antigen of interest can be measuredusing any method known to a person skilled in the art. For instance, anantigen of interest is labeled with, for example, a fluorescent label.Detection of binding can subsequently be determined by varioustechniques, among which fluoresce microscopy and Fluorescence ActivatedCell Sorting (FACS). FACS allows separation of cells in a suspension onthe basis of size and the fluorescence of conjugated antibodies directedagainst surface antigens.

Selecting at least one high-affinity B-cell from a population ofB-cells, preferably from a monoclonal B-cell line, can be performedusing any method known to a person skilled in the art. Selection of atleast one high-affinity B-cell hereof is, for instance, performed bycell sorting, for instance, using FACS (see above) or limited dilution.Limited dilution comprises the serial dilution of a suspension of cells,for instance, B-cells, until a single cell is present in a given volume.Subsequently, the binding capacity of each B-cell (after expansion ofsingle cells to a population) is tested to allow selection of aB-cell-producing antibodies with a high affinity for antigen.

A B-cell capable of producing antibody is defined as a B-cell that iscapable of producing and/or secreting antibody or a functional partthereof, and/or that is capable of developing into a cell that iscapable of producing and/or secreting antibody or a functional partthereof.

A functional part of an antibody is defined as a part that has at leastone same property as the antibody in kind, not necessarily in amount.The functional part is preferably capable of binding a same antigen asthe antibody, albeit not necessarily to the same extent. A functionalpart of an antibody preferably comprises a single domain antibody, asingle chain antibody, a FAB fragment, a nanobody, a unibody, a singlechain variable fragment (scFv), or a F(ab′)2 fragment.

Non-limiting examples of a B-cell used or selected in a method hereofinclude B-cells derived from a human individual, rodent, rabbit, llama,pig, cow, goat, horse, ape, chimpanzee, macaque and gorilla. Preferably,the B-cell is a human cell, a murine cell, a rabbit cell, an ape cell, achimpanzee cell, a macaque cell and/or a llama cell. Most preferably,the B-cell is a human B-cell.

In a preferred embodiment, a memory B-cell is selected in step a) of themethod as described herein, for instance, a human memory B-cell. In aparticularly preferred embodiment, the memory B-cell is a peripheralblood memory B-cell. Peripheral blood memory B-cells are easilyobtained, without much discomfort for the individual from which they arederived, and appear to be very suitable for use in a method according tothe instant disclosure.

A B-cell or a population of B-cells, preferably a monoclonal B-cellline, obtained with a method hereof is preferably stable for at leastfour weeks, more preferably at least six weeks, more preferably at leastnine weeks, more preferably for at least three months, more preferablyfor at least six months, meaning that such B-cells are capable of bothreplicating and producing antibody, or capable of replicating anddeveloping into a cell that produces antibody, during the time periods.B-cells hereof preferably comprise cells producing IgM or cellsproducing other immunoglobulin isotypes like IgG, or IgA, or IgE,preferably IgG. A B-cell hereof is particularly suitable for use inproducing an antibody-producing cell line. High-affinity B-cells or apopulation or monoclonal B-cell line of high-affinity B-cells hereof arepreferably cultured ex vivo and antibody is preferably collected forfurther use. Antibodies or functional parts thereof produced with amethod hereof are useful for a wide variety of applications, such as,for instance, therapeutic, prophylactic and diagnostic applications, aswell as research purposes and ex vivo experiments. For instance, ascreening assay is performed wherein antibodies or functional partshereof are incubated with a sample in order to determine whether anantigen of interest is present.

In one embodiment, a high-affinity B-cell or a population or monoclonalB-cell line of high-affinity B-cells hereof comprises a human B-cell,capable of producing human antibody, because human antibodies areparticularly suitable for therapeutic and/or prophylactic applicationsin human individuals.

The expression of BCL6 in a B-cell is induced, enhanced and/ormaintained in a variety of ways. In one embodiment, a B-cell is providedwith a nucleic acid encoding BCL6 or a homologue. In another embodiment,a B-cell is provided with a compound capable of directly or indirectlyenhancing BCL6 expression. Such compound preferably comprises a SignalTransducer of Activation and Transcription 5 (STAT5) protein or afunctional part, derivative and/or analogue thereof, and/or a nucleicacid sequence coding therefor. STAT5 is a signal transducer capable ofenhancing BCL6 expression. There are two known forms of STAT5, STAT5aand STAT5b that are encoded by two different, tandemly linked genes.Administration and/or activation of STAT5 results in enhanced BCL6levels. Hence, down-regulation of BCL6 by BLIMP1 is at least in partcompensated by up-regulation of expression of BCL6 by STAT5 or afunctional part, derivative and/or analogue thereof. Hence, STAT5 or afunctional part, derivative and/or analogue thereof is capable ofdirectly influencing BCL6 expression. It is also possible to indirectlyinfluence BCL6 expression. This is, for instance, done by regulating theamount of a compound that, in turn, is capable of directly or indirectlyactivating STAT5 and/or regulating STAT5 expression. Hence, in oneembodiment, the expression and/or activity of endogenous and/orexogenous STAT5 is increased. It is, for instance, possible toindirectly enhance BCL6 expression by culturing an antibody-producingcell in the presence of interleukin (IL) 2 and/or IL 4 or othercytokines that are capable of activating STAT5.

It is furthermore preferred that in a method hereof, the B-cells are atleast at some stage incubated with IL 21 and CD40L. A B-cell, such as anantibody-producing plasmablast-like B-cell, is preferably cultured inthe presence of CD40L since replication of most B-cells is favored byCD40L. It is furthermore preferred that STAT3 is activated in theB-cell. Most preferably, IL 21 is used for up-regulating STAT3, since IL21 is particularly suitable for influencing the stability of a B-cellhereof. In addition to up-regulating STAT3, IL 21 is capable ofup-regulating BLIMP1 expression even when BLIMP1 expression iscounteracted by BCL6.

In another embodiment, the amount of BLIMP1 expression product in theB-cell selected in step a) of a method hereof is directly or indirectlycontrolled. The amount of BLIMP1 expression product can be controlled invarious ways, for instance, by regulating STAT3 or a functional part,derivative or analogue thereof. STAT3 is activated in a variety of ways.Preferably, STAT3 is activated by providing a B-cell hereof with acytokine. Cytokines, being naturally involved in B-cell differentiation,are very effective in regulating STAT proteins. Very effectiveactivators of STAT3 are IL-21 and IL-6, but also IL-2, IL-7, IL-10,IL-15 and IL-27 are known to activate STAT3. Moreover, Toll-likereceptors (TLRs) that are involved in innate immunity are also capableof activating STAT3. Most preferably, IL-21 is used. IL-21 is capable ofup-regulating BLIMP1 expression even when BLIMP1 expression iscounteracted by BCL6.

By “a functional part of STAT5 or STAT3” is meant a proteinaceousmolecule that has the same capability—in kind, not necessarily inamount—of influencing the stability of an antibody-producing cell ascompared to STAT5 or STAT3, respectively. A functional part of a STAT5protein or a STAT3 protein is, for instance, devoid of amino acids thatare not, or only very little, involved in said capability. A derivativeof STAT5 or STAT3 is defined as a protein that has been altered suchthat the capability of the protein of influencing the stability of anantibody-producing cell is essentially the same in kind, not necessarilyin amount. A derivative is provided in many ways, for instance, throughconservative amino acid substitution wherein one amino acid issubstituted by another amino acid with generally similar properties(size, hydrophobicity, etc.), such that the overall functioning islikely not to be seriously affected. A derivative, for instance,comprises a fusion protein, such as a STAT5-ER fusion protein whoseactivity depends on the presence of 4 hydroxy-tamoxifen (4HT). Ananalogue of STAT5 or STAT3 is defined as a molecule having the samecapability of influencing the stability of an antibody-producing cell inkind, not necessarily in amount. The analogue is not necessarily derivedfrom the STAT5 or STAT3 protein.

A method hereof is preferably used for generating a cell line ofhigh-affinity B-cells that is stable for at least one week, preferablyat least one month, more preferably at least three months, morepreferably at least six months so that commercial high-affinity antibodyproduction has become possible. Preferably, a stable cell line capableof producing monoclonal high-affinity antibodies is produced. This ispreferably performed by using memory B-cells that have, for instance,been isolated from a sample by selection for CD19 (B-cell marker) andcell surface IgG and/or CD27 (to mark memory cells). Furthermore, amemory B-cell capable of specifically binding an antigen of interest is,for instance, selected in a binding assay using the antigen of interest.Subsequently, BCL6 and an anti-apoptotic nucleic acid, preferably Bcl-XLor Mcl-1, are preferably co-expressed in the B-cell, resulting in apopulation of cells specific for the antigen of interest. Preferably,only one memory cell is selected in step a) of a method as describedherein, so that a B-cell population hereof producing monoclonalantibodies (a monoclonal B-cell line) is obtained.

In one embodiment, a B-cell, preferably, but not necessarily, a memoryB-cell, that originates from an individual that had been previouslyexposed to an antigen of interest, is used in a method hereof. However,this is not necessary. It is also possible to use a B-cell from anindividual that has not been exposed to the antigen of interest. Forinstance, a B-cell is used that is specific for another antigen butshows cross-reactivity with the antigen of interest. As another example,a B-cell is used that is selected from a naïve B-cell population of anindividual. The naïve B-cell population of an individual may containB-cells that show reactivity with an antigen of interest even though theindividual has not been exposed to the antigen of interest. Such B-cellfrom a naïve B-cell population is, for instance, selected using labeledantigen of interest.

Furthermore provided are isolated or recombinant B-cells and populationsof B-cells, preferably monoclonal B-cell lines, obtained by a methodhereof. Such high-affinity B-cells are preferably stable for at leastone week, preferably for at least one month, more preferably for atleast three months, more preferably for at least six months, meaningthat the B-cell is capable of both replicating and producing antibody,or capable of replicating and developing into a cell that producesantibody, during the time periods. B-cells hereof preferably comprisecells producing IgM or cells producing other immunoglobulin isotypeslike IgG, or IgA, or IgE, preferably IgG. A B-cell hereof isparticularly suitable for use in producing an antibody-producing cellline. High-affinity B-cells hereof are preferably cultured ex vivo andantibody is preferably collected for further use. Antibodies obtainedfrom a B-cell or from a B-cell population or monoclonal cell line hereofare also provided. High-affinity antibodies or functional parts thereofproduced with a method hereof are useful for a wide variety ofapplications, such as, for instance, therapeutic, prophylactic anddiagnostic applications, as well as research purposes and ex vivoexperiments. For instance, a screening assay is performed whereinantibodies or functional parts hereof are incubated with a sample inorder to determine whether an antigen of interest is present.

B-cells generated with a method hereof are particularly suitable forproducing high-affinity antibodies against an antigen of interest. Inone preferred embodiment, however, the genes encoding the Ig heavyand/or light chains are isolated from the cell and expressed in a secondcell, such as, for instance, cells of a Chinese hamster ovary (CHO) cellline. The second cell, also called herein a “producer cell,” ispreferably adapted to commercial antibody production. Proliferation ofthe producer cell results in a producer cell line capable of producingantibody. Preferably, the producer cell line is suitable for producingcompounds for use in humans. Hence, the producer cell line is preferablyfree of pathogenic agents such as pathogenic micro-organisms.

The disclosure is further explained by the following, non-limiting,examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Panel A: Binding of labeled HA H3 protein to the BCR ofH3-specific cells within a polyclonal B-cell population. B-cells thatbind the H3 protein with high affinity were cloned by single cellsorting. After two to three weeks of culture, the culture supernatantwas screened for H3-specific antibodies. Panel B: Example of thescreening performed to select H3-specific clones. Left panel: Screeningby ELISA. Recombinant H3 protein was coated onto a plate followed byincubation with culture supernatant. Antibody binding was detected usinganti-human-IgG-HRP. Right panel: Screening on cell surface-expressed HA.H3N2-infected cells were incubated with B-cell culture supernatant.Antibody binding was detected with a PE-labeled goat anti-human F(ab′)2.

FIG. 2: Left panel: mRNA levels of AICDA in CD19+CD38+CD20+IgD-tonsillarGC B-cells and CD19+IgG+CD27+ peripheral blood memory B-cells comparedto 23 BCL6- and Bcl-xL-transduced monoclonal cell lines. Right panel:Selection of high or low binding subclones within an H3-specific clone.Boxed populations were selected by cell sorting and further expanded.

FIG. 3: Panel A: FACS analysis for the binding of labeled HA H3 toselected cells 13 days after their selection for higher or lower H3 BCRbinding from a clonal cell. Increased or lowered H3 binding ismaintained and stable after subcloning. Panel B: FACS staining for theBCR of the different selected subpopulations. Increased or loweredlevels of H3 binding to selected populations correlates with the BCRexpression on the cell surface of these populations. Light grey line:B-cells selected for high H3 binding; filled graph: B-cells not selected(parental cells); dark grey line: B-cells selected for low H3 binding.

FIG. 4: H3 ELISA of the culture supernatant of the different(sub)populations. Secreted IgG from cells that were selected for higherbinding to H3-APC protein show increased binding in the H3 ELISAcompared to IgG from the non-sorted parental line. Top line: B-cellsselected for high H3 binding; middle line: B-cells not selected(parental cells); bottom line: B-cells selected for low H3 binding.

FIG. 5: Selection of high- or low-affinity subclones within anH3-specific clone (AT10_(—)004). Cells were stained with Alexa-647labeled HA H3 antigen together with IgG-PE antibody. Circled populationswere selected by cell sorting and further expanded.

FIG. 6: FACS analysis for the binding of labeled HA H3 together with aBCR stain (either for the heavy chain, IgG-PE, or for the light chain,Kappa-PE) to selected cells two weeks after the third selection roundfor higher or lower H3 BCR binding and to the parental AT10_(—)004.

FIG. 7: Overview of the amino acid changes that were found in theselected subpopulations with increased or decreased affinity. Mutationsin the sequence of AT10_(—)004 that were associated with increased H3antigen binding were incorporated in the AT10_(—)004 sequence and theseantibodies were produced recombinant in 239T cells and purified forfurther analysis (AT10_(—)004 mutant B and AT10_(—)004 mutant C).

FIG. 8: SPR analysis of the binding of AT10_(—)004 antibodies to HA H3.Association curves of antibodies AT10_(—)004, AT10_(—)004 mutant A,AT10_(—)004 mutant B and AT10_(—)004 mutant C.

FIG. 9: Mean fluorescent intensity (MFI) of AT10_(—)004 antibodyvariants binding to H3N2-infected cells in a FACS assay. Differentconcentrations of recombinant AT10_(—)004, AT10_(—)004 mutant B,AT10_(—)004 mutant C and Rituximab (negative control) were incubatedwith H3N2-infected cells. Antibody binding was detected with PE-labeledgoat anti-human F(ab′) 2. Plotted is the mean and the SEM of the MFI ofthe resulting PE signal.

DETAILED DESCRIPTION Examples Example 1 Generation of an Anti-InfluenzaHemaglutinin (HA) H3-Specific Monoclonal Human Antibody

Human memory B-cells were immortalized using the BCL6/Bcl-xL technologydescribed by Kwakkenbos et al. (Generation of stable monoclonalantibody-producing B-cell receptor-positive human memory B-cells bygenetic programming, Nature Medicine (2010) vol. 16 (1) pp. 123-8, andpatent application MEANS AND METHODS FOR INFLUENCING THE STABILITY OFANTIBODY PRODUCING CELLS [WO 2007/067046]). In brief, BCL6 andBcl-xL-transduced cells (GFP-positive) were cultured withCD40Ligand-expressing L-cells and interleukin (IL)-21 before the HA H3binding cells were sorted using the Fluorescence activated cell sorter(FACS) (FIG. 1A). The Influenza HA protein (Protein Sciences) waslabeled with Alexa Fluor 647 (Molecular Probes) and incubated withpolyclonal cultured B-cells. HA-positive cells were sorted single cellper well and maintained in culture for two to three weeks before theclones were screened for HA binding by 1) ELISA or 2) binding toH3-infected cells (FIG. 1, Panel B).

Example 2 Selection of a Higher- and Lower-Affinity B-Cell Clone

Since the BCL6 Bcl-XL-transduced B-cells express the enzyme ActivationInduced Deaminase (AID, gene nomenclature is AICDA) as described byKwakkenbos et al. (FIG. 2, left panel, and “Generation of stablemonoclonal antibody-producing B-cell receptor-positive human memoryB-cells by genetic programming,” Nature Medicine (2010) vol. 16 (1) pp.123-8) an individual B-cell can make nucleotide changes in theimmunoglobulin heavy and light chains. These changes may influence thebinding affinity of the clones to its antigen. To determine if subclonesof the HA H3 binding clone indeed can have a different binding profile,the H3-specific clone was again incubated with labeled HA H3 antigen.Using the FACS, a population of high HA H3 binding cells and apopulation of low HA H3 binding cells were sorted (FIG. 2, right panel)and maintained in culture for at least 13 days before the B-cellsupernatant was harvested and tested.

Example 3 HA H3 High- and Low-Affinity Sorted B-Cells Express a Stablebut Variable Level of Surface Immunoglobulin

First, we characterized the stability of the sorted cells by analyzingthe binding capacity of the B-cell receptor (BCR) to labeled HA H3 byFACS (FIG. 3, Panel A). Since the HA H3 high sorted cells still showedhigher binding abilities, we next determined the surface immunoglobulinexpression level by FACS (FIG. 3, Panel B). It was observed that thecells sorted for a relatively low binding capacity to HA H3 did expressless immunoglobulin protein on the surface compared to cells sorted forhigh HA binding. This higher or lower BCR expression and BCR binding toHA H3 protein was maintained over time and became even more pronouncedafter a second round of sorting (data not shown).

Example 4 Affinity for HA H3 of the Antibodies Derived from the Originaland High- and Low-Affinity HA H3 Binding Cells

To determine the binding affinity of the antibodies produced by thedifferent HA H3-recognizing B-cells, the culture supernatant of the day13 cultures of the original HA H3 binding cells and of the high- andlow-affinity HA H3 binding cells was analyzed by ELISA. HA H3 (1 μg/mlProtein Sciences) was coated directly on the plate before the wells wereincubated with the different B-cell supernatants. Binding of the humanIgGs to the HA H3 protein was detected with an anti-human polyclonalgoat antibody that was HRP labeled. Secreted IgG from cells that wereselected for higher binding to H3-APC protein show increased binding inthe H3 ELISA compared to IgG from the non-sorted parental line (FIG. 4).

Example 5 Combined BCR-Antigen Stain for the Selection of High- andLow-Affinity Clones

In Examples 2 and 3, it is shown that selection of B-cells, within theheterogeneous subpopulation of a monoclonal B-cell clone, with thehighest level of H3 binding may select for cells that have elevatedlevels of BCR expression. Thus, when selection is done solely based onthe level of H3 binding, cells that have increased antigen affinity butlow levels of BCR expression might be excluded. To exclude the influenceof the level of immunoglobulin expression on the selection ofhigh-affinity clones, a new set of selection rounds were performed usinga combination of antigen staining (H3-Alexa-647) and BCR staining (FIG.5). BCR staining was performed with antibodies that bind to the heavy orthe light chain of the BCR. High H3 staining and low BCR stainingindicates high antigen affinity, whereas low H3 staining and high BCRstaining indicates low antigen affinity.

An HA H3-specific B-cell clone (AT10_(—)004) was cultured for two tothree weeks to produce millions of cells before an antigen-BCR stainingwas performed. Cells that showed deviating antigen affinity, bothpositive and negative, were selected and sorted on a cell sorter. Afterthree rounds of sorting and growing, FACS analysis was performed onthese cells to determine differences in antigen binding. Cells that weresorted three times for increased or decreased antigen binding show aclear shift in antigen binding compared to non-selected cells (FIG. 6).FIG. 6 demonstrates that increased or lowered H3 binding is maintainedand stable after selection.

Example 6 Sequencing of the BCR from Selected Cells

We isolated total RNA with the RNEAsY® mini kit (Qiagen) fromAT10_(—)004 and AT10_(—)004 mutant B-cell cultures selected for high orlow affinity, generated cDNA from the RNA, performed PCR and analyzedthe sequence of the heavy chain and light chain of the BCR. A mutationleading to an amino acid change at position 38 (CDR1), resulting in theexchange of the Glycine to an Alanine in the heavy chain, was found forthe cells that were sorted for decreased affinity (hereafter named“mutant A”). Mutations leading to amino acid changes in the light chain(compared to the parental AT10_(—)004 sequence) were found for theincreased affinity sorted cells. Sequence analysis showed a change ofamino acid 108 (CDR3) in the kappa light chain from a Serine to aTyrosine (hereafter named “mutant B”). An additional mutation atposition 38 leading to replacement of Tyrosine to a Phenylalanine wasfound in some sequences (hereafter named “mutant C”) (FIG. 7 and Table1). To produce recombinant AT10_(—)004 and increased affinity, mutants Band C mAb, we cloned the heavy and light variable regions in frame withhuman IgG1 and Kappa constant regions into a pcDNA3.1 (Invitrogen)-basedvector and transiently transfected 293T cells. We purified recombinantmAb from the culture supernatant with an AKTA (GE healthcare).

Example 7 Surface Plasmon Resonance (SPR) Analysis

SPR analysis was performed on an IBIS MX96 SPR imaging system (IBISTechnologies BV., Enschede, The Netherlands) as described (Lokate etal., 2007, J. Am. Chem. Soc. 129:14013-140318). In short, one SPRanalysis cycle consists of one or more incubation steps in whichanalytes are flushed over a coated sensor, followed by a regenerationstep, in which any bound analyte is removed from the sensor. Multiplecycles can be performed in one experiment.

Dilution series (concentration ranging from 0.30 to 10 μg/ml) ofAT10_(—)004 and AT10_(—)004 mutant antibody in coupling buffer(PBS+0.03% TWEEN® 20+0.01 mg/ml BSA) were immobilized during 99 minuteson a human-IgG-specific gold-film gel-type SPR-chip (Ssens, Enschede,The Netherlands) using a continuous flow microspotter device (WasatchMicrofluidics, Salt Lake City, Utah, USA). After spotting, the sensorwas washed three times with PBS+0.03% TWEEN® 20 (PBST).

To block any unoccupied sites in the anti-IgG coated SPR chip, the chipwas first injected with a non-specific human IgG (rituximab, 10 μg/ml inPBST) and incubated for 45 minutes, followed by 100 minutes incubationwith PBST. After this blocking step, two blank injection cycles weredone, each consisting of 45 minutes injection with empty assay buffer(1× PBST+0.01% BSA) followed by 100 minutes incubation with PBST. Then,the sensor was injected with 1 μg/ml recombinant influenza HA3-protein(from H3N2, Wyoming, March 2003, Sino Biological Inc., Beijing, P.R.China) in assay buffer and incubated for 45 minutes. Subsequently, thesensor is washed with PBST and incubated for 100 minutes (to measurecomplex dissociation). Obtained data was analyzed using Sprint software(version 1.6.8.0, IBIS Technologies BV., Enschede, The Netherlands).Binding constants were fitted using Scrubber2 software (BiologicSoftware, Campbell, Australia). FIG. 8 shows that recombinant HA3 doesnot associate with AT10_(—)004 mutant A. An increased association rateof HA3 for AT10_(—)004 mutants B and C is seen compared to thenon-mutated AT10_(—)004. The binding constants obtained for AT10_(—)004and each mutant are shown in Table 2.

Example 8 Antibody Binding to Virus-Infected Cells

To test the binding capacity of AT10_(—)004 and the AT10_(—)004 mutantsto virus-infected cells, we performed FACS analysis on Influenza H3N2(A/Netherlands/177/2008)-infected cells. MDCK-SIAT cells were grown in aT175 culture flask to 80-100% confluency in DMEM/FCS/PS/G418. The celllayer was washed 2× with 10 ml PBS after which 15 ml ofOptimem/PS/G418/Trypsin was added. Subsequently, 0.5 ml of 100,000TCID50 Influenza virus was added to the flask and cells were cultured at37° C. After 24-48 hours, the cells were washed 2× with 10 ml PBS anddetached from the plastic using Trypsin-EDTA. Cells were counted andfrozen at −150° C. until use. The infected cells were defrosted andincubated with AT10_(—)004 (mutant) antibodies or Rituximab (as negativecontrol) at several concentrations for 30 minutes at 4° C. and thenwashed 2× with 150 μl PBS/2% FCS. Antibody binding was detected withanti-human IgG-PE (Southern Biotech) and analyzed on a Guava easyCyteBHT, Millipore). AT10_(—)004 mutants B and C both show increasedstaining intensity on H3N2-infected cells compared to the parentalAT10_(—)004 antibody (FIG. 9).

TABLE 1Amino acid and nucleotide sequences of antibodies AT10-004 and AT10-004 mutantsA, B and C. In the mutant sequences, mutations as compared to antibodyAT10-004 are indicated in bold and underlined SEQ ID NO AntibodyIdentity Sequence 1 AT10_004 Heavy chain RHGIS CDR1 2 AT10_004Heavy chain WISAYTGDTDYAQKFQG CDR2 3 AT10_004 Heavy chainLRLQGEVVVPPSQSNWFDP CDR3 4 AT10_004 Light chain RASQSVSRYLA CDR1 5AT10_004 Light chain DASNRAT CDR2 6 AT10_004 Light chain QQRSNWLK CDR3 7AT10_004 Heavy chainQVQLVQSGAEVRKPGASVKVSCKASGYTFTRHGISWVRQAPGQGLEWMGWISAYTGDTDYAQKFQGRVTMTTDTSTNTAYMELRSLRSDDAAVYYCARLRLQGEVVVPPSQSNW FDPWGQGTLVTVSS8 AT10_004 Light chainEIVLTQSPATLSLYPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWLKITFGQGTRLEIKGTV 9 AT10_004Heavy chain agg cat ggt atc agc CDR1 10 AT10_004 Heavy chaintgg atc agc get tac act ggt gac aca gac tat gca cag aaa ttc cag ggg CDR211 AT10_004 Heavy chainctt cgt ttg cag ggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttc CDR3gac ccc 12 AT10_004 Light chainagg gcc agt cag agt gtt agc agg tac tta gcc CDR1 13 AT10_004 Light chaingat gca tcc aac agg gcc act CDR2 14 AT10_004 Light chaincag cag cgt gac aac tgg ctt aag CDR3 15 AT10_004 Heavy chaincag gtt cag ctg gtg cag tct gga gct gag gtg agg aag cct ggg gcc tcagtg aag gtc tcc tgc aag gct tcc ggt tac acg ttt acc agg cat ggt atcagc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg gga tgg atcagc gct tac act ggt gac aca gac tat gca cag aaa ttc cag ggg cga gtcacc atg acc aca gat aca tcc acg aac aca gcc tac atg gaa ctg agg agcctg aga tct gac gac gcg gcc gta tat tac tgt gcg aga ctt cgt ttg cagggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttc gac ccc tgg ggccag gga acc ctg gtc acc gtc tcc tca 16 AT10_004 Light chaingaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tat cca ggg gaaaga gcc acc ctc tct tgc agg gcc agt cag agt gtt agc agg tac tta gcctgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gcatcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc agt ggg tct gggaca gac ttc acc ctc acc atc agc agc cta gag cct gaa gat ttt gca gtttat tac tgt cag cag cgt gac aac tgg ctt aag atc acc ttc ggc caa gggaca cga ctg gaa att aaa gga act gtg 17 AT10_004 Heavy chain RH A ISmutant A CDR1 18 AT10_004 Heavy chain WISAYTGDTDYAQKFQG mutant A CDR2 19AT10_004 Heavy chain LRLQGEVVVPPSQSNWFDP mutant A CDR3 20 AT10_004Light chain RASQSVSRYLA mutant A CDR1 21 AT10_004 Light chain DASNRATmutant A CDR2 22 AT10_004 Light chain QQRSNWLK mutant A CDR3 23 AT10_004Heavy chain QVQLVQSGAEVRKPGASVKVSCKASGYTFTRH A ISWVRQAPGQGLEWMGWISAYTGDmutant A TDYAQKFQGRVTMTTDTSTNTAYMELRSLRSDDAAVYYCARLRLQGEVVVPPSQSNWFDPWGQGTLVTVSS 24 AT10_004 Light chainEIVLTQSPATLSLYPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPA mutant ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWLKITFGQGTRLEIKGTV 25 AT10_004Heavy chain agg cat g c t atc agc mutant A CDR1 26 AT10_004 Heavy chaintgg atc agc gct tac act ggt gac aca gac tat gca cag aaa ttc cag gggmutant A CDR2 27 AT10_004 Heavy chainctt cgt ttg cag ggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg mutant ACDR3 ttc gac ccc 28 AT10_004 Light chainagg gcc agt cag agt gtt agc agg tac tta gcc mutant A CDR1 29 AT10_004Light chain gat gca tcc aac agg gcc act mutant A CDR2 30 AT10_004Light chain cag cag cgt agc aac tgg ctt aag mutant A CDR3 31 AT10_004Heavy chaincag gtt cag ctg gtg cag tct gga gct gag gtg agg aag cct ggg gcc tcamutant Agtg aag gtc tcc tgc aag gct tcc ggt tac acg ttt acc agg cat gct atcagc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg gga tgg atcagc gct tac act ggt gac aca gac tat gca cag aaa ttc cag ggg cga gtcacc atg acc aca gat aca tcc acg aac aca gcc tac atg gaa ctg agg agcctg aga tct gac gac gcg gcc gta tat tac tgt gcg aga ctt cgt ttg cagggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttc gac ccc tgg ggccag gga acc ctg gtc acc gtc tcc tca 32 AT10_004 Light chaingaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tat cca ggg gaamutant Aaga gcc acc ctc tct tgc agg gcc agt cag agt gtt agc agg tac tta gcctgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gcatcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc agt ggg tct gggaca gac ttc acc ctc acc atc agc agc cta gag cct gaa gat ttt gca gtttat tac tgt cag cag cgt agc aac tgg ctt aag atc acc ttc ggc caa gggaca cga ctg gaa att aaa gga act gtg 33 AT10_004 Heavy chain RHGISmutant B CDR1 34 AT10_004 Heavy chain WISAYTGDTDYAQKFQG mutant B CDR2 35AT10_004 Heavy chain LRLQGEVVVPPSQSNWFDP mutant B CDR3 36 AT10_004Light chain RASQSVSRYLA mutant B CDR1 37 AT10_004 Light chain DASNRATmutant B CDR2 38 AT10_004 Light chain QQR Y NWLK mutant B CDR3 39AT10_004 Heavy chainQVQLVQSGAEVRKPGASVKVSCKASGYTFTRHGISWVRQAPGQGLEWMGWISAYTGD mutant BTDYAQKFQGRVTMTTDTSTNTAYMELRSLRSDDAAVYYCARLRLQGEVVVPPSQSNW FDPWGQGTLVTVSS40 AT10_004 Light chainEIVLTQSPATLSLYPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPA mutant BRFSGSGSGTDFTLTISSLEPEDFAVYYCQQR Y NWLKITFGQGTRLEIKGTV 41 AT10_004Heavy chain agg cat ggt atc agc mutant B CDR1 42 AT10_004 Heavy chaintgg atc agc gct tac act ggt gac aca gac tat gca cag aaa ttc cag gggmutant B CDR2 43 AT10_004 Heavy chainctt cgt ttg cag ggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttcmutant B CDR3 gac ccc 44 AT10_004 Light chainagg gcc agt cag agt gtt agc agg tac tta gcc mutant B CDR1 45 AT10_004Light chain gat gca tcc aac agg gcc act mutant B CDR2 46 AT10_004Light chain cag cag cgt  ta c aac tgg ctt aag mutant B CDR3 47 AT10_004Heavy chaincag gtt cag ctg gtg cag tct gga gct gag gtg agg aag cct ggg gcc tcamutant Bgtg aag gtc tcc tgc aag gct tcc ggt tac acg ttt acc agg cat ggt atcagc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg gga tgg atcagc gct tac act ggt gac aca gac tat gca cag aaa ttc cag ggg cga gtcacc atg acc aca gat aca tcc acg aac aca gcc tac atg gaa ctg agg agcctg aga tct gac gac gcg gcc gta tat tac tgt gcg aga ctt cgt ttg cagggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttc gac ccc tgg ggccag gga acc ctg gtc acc gtc tcc tca 48 AT10_004 Light chaingaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tat cca ggg gaamutant Baga gcc acc ctc tct tgc agg gcc agt cag agt gtt agc agg tac tta gcctgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gcatcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc agt ggg tct gggaca gac ttc acc ctc acc atc agc agc cta gag cct gaa gat ttt gca gtttat tac tgt cag cag cgt  ta c aac tgg ctt aag atc acc ttc ggc caa gggaca cga ctg gaa att aaa gga act gtg 49 AT10_004 Heavy chain RHGISmutant C CDR1 50 AT10_004 Heavy chain WISAYTGDTDYAQKFQG mutant C CDR2 51AT10_004 Heavy chain LRLQGEVVVPPSQSNWFDP mutant C CDR3 52 AT10_004Light chain RASQSVSR F LA mutant C CDR1 53 AT10_004 Light chain DASNRATmutant C CDR2 54 AT10_004 Light chain QQR Y NWLK mutant C CDR3 55AT10_004 Heavy chainQVQLVQSGAEVRKPGASVKVSCKASGYTFTRHGISWVRQAPGQGLEWMGWISAYTGD mutant CTDYAQKFQGRVTMTTDTSTNTAYMELRSLRSDDAAVYYCARLRLQGEVVVPPSQSNW FDPWGQGTLVTVSS56 AT10_004 Light chain EIVLTQSPATLSLYPGERATLSCRASQSVSR FLAWYQQKPGQAPRLLIYDASNRATGIPA mutant C RFSGSGSGTDFTLTISSLEPEDFAVYYCQQR YNWLKITFGQGTRLEIKGTV 57 AT10_004 Heavy chain agg cat ggt ate agc mutant CCDR1 58 AT10_004 Heavy chaintgg atc agc gct tac act ggt gac aca gac tat gca cag aaa ttc cag gggmutant C CDR2 59 AT10_004 Heavy chainctt cgt ttg cag ggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttcmutant C CDR3 gac ccc 60 AT10_004 Light chainagg gcc agt cag agt gtt agc agg t t c tta gcc mutant C CDR1 61 AT10_004Light chain gat gca tcc aac agg gcc act mutant C CDR2 62 AT10_004Light chain cag cag cgt  ta c aac tgg ctt aag mutant C CDR3 63 AT10_004Heavy chaincag gtt cag ctg gtg cag tct gga get gag gtg agg aag cct ggg gcc tcamutant Cgtg aag gtc tcc tgc aag gct tcc ggt tac acg ttt acc agg cat ggt atcagc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg gga tgg atcagc gct tac act ggt gac aca gac tat gca cag aaa ttc cag ggg cga gtcacc atg acc aca gat aca tcc acg aac aca gcc tac atg gaa ctg agg agcctg aga tct gac gac gcg gcc gta tat tac tgt gcg aga ctt cgt ttg cagggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttc gac ccc tgg ggccag gga acc ctg gtc acc gtc tcc tca 64 AT10_004 Light chaingaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tat cca ggg gaamutant C aga gcc acc ctc tct tgc agg gcc agt cag agt gtt agc agg t tc tta gcctgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gcatcc aac agg gcc act ggc ate cca gcc agg ttc agt ggc agt ggg tct gggaca gac ttc acc ctc acc atc agc agc cta gag cct gaa gat ttt gca gtttat tac tgt cag cag cgt  ta c aac tgg ctt aag atc acc ttc ggc caa gggaca cga ctg gaa att aaa gga act gtg

TABLE 2 Binding constants for AT10-004 and mutants Antibody: k_(a):k_(d): K_(D): AT10-004 1.4 (±0.1) 0.1 70 (±10) AT10-004, mutant A 0 — —AT10-004, mutant B 1.9 (±0.1) 0.1 50 (±10) AT10-004, mutant C 1.7 (±0.1)0.1 60 (±10) k_(a) in 10⁴ sec⁻¹ * M⁻¹, k_(d) in 10⁻⁵ sec⁻¹, K_(D) in pMConstants were fitted in Scrubber2, using a global fit to a 1:1interaction model.

The invention claimed is:
 1. A method for producing high affinityantibodies specific for an antigen of interest, the method comprisingthe steps of: a) selecting a B-cell able to produce antibody specificfor said antigen of interest or selecting a B-cell able to differentiateinto a B-cell that is able to produce antibody specific for said antigenof interest; b) inducing, enhancing and/or maintaining expression ofBCL6 in said B-cell; c) inducing, enhancing and/or maintainingexpression of an anti-apoptotic nucleic acid in said B-cell; d) allowingexpansion of said B-cells into a first population of B-cells; e)performing antigen staining and BCR staining of B-cells from said firstpopulation; f) selecting a B-cell that has a high antigen affinity; g)allowing expansion of said B-cell into a second population of B-cells;h) measuring the antigen association rate and dissociation rate ofantibodies produced by at least one B-cell from said second population;and i) selecting an antibody with an affinity for said antigen ofinterest that is higher than the average affinity of said firstpopulation of B-cells for said antigen of interest.
 2. The methodaccording to claim 1, further comprising, following step g), repeatingsteps e), f) and g) at least once.
 3. The method according to claim 1,wherein said at least one B-cell is cultured for at least four weeks. 4.The method according to claim 1, wherein said B-cell selected in step a)is a memory B-cell.
 5. The method according to claim 1, wherein saidanti-apoptotic nucleic acid comprises a gene encoding an anti-apoptoticmolecule.
 6. The method according to claim 1, further comprising:providing said B-cell with a growth factor.
 7. The method according toclaim 1, further comprising: directly or indirectly controlling theamount of BLIMP1 expression product in said B-cell selected in step a).8. The method according to claim 1, wherein said B-cell selected in stepa) originates from an individual that had been previously exposed tosaid antigen of interest.
 9. The method according to claim 1, furthercomprising: expressing a gene derived of said at least one B-cellencoding the Ig heavy chain and/or Ig light chain in a second cell. 10.The method according to claim 5, wherein said B-cell selected in step a)is a human memory B-cell.
 11. The method according to claim 6, whereinthe anti-apoptotic nucleic acid comprises a gene encoding ananti-apoptotic molecule of the BCL2 family.
 12. The method according toclaim 6, wherein the anti-apoptotic nucleic acid comprises a geneencoding Bcl-xL or a functional part thereof.
 13. The method accordingto claim 6, wherein the anti-apoptotic nucleic acid comprises a geneencoding Mcl-1, or a functional part thereof.
 14. The method accordingto claim 7, wherein the growth factor is IL21.
 15. The method accordingto claim 7, wherein the growth factor is CD40L.
 16. A method forproducing high affinity antibodies specific for an antigen of interest,the method comprising the steps of: a) selecting a B-cell able toproduce antibody specific for the antigen of interest or selecting aB-cell able to differentiate into a B-cell that is able to produceantibody specific for the antigen of interest, wherein the B-cell is ahuman memory B-cell that originates from an individual who haspreviously been exposed to the antigen of interest; b) inducing,enhancing and/or maintaining expression of BCL6 in the selected B-cell;c) inducing, enhancing and/or maintaining expression of ananti-apoptotic nucleic acid in the B-cell, wherein the anti-apoptoticnucleic acid comprises a gene encoding an anti-apoptotic molecule of theBCL2 family; d) allowing expansion of said B-cells into a firstpopulation of B-cells; e) performing antigen staining and BCR stainingof B-cells from said first population; f) selecting a B-cell that has ahigh antigen affinity; g) allowing expansion of the B-cell into a secondpopulation of B-cells; h) measuring the antigen association rate anddissociation rate of antibodies produced by at least one B-cell from thesecond population; and i) selecting an antibody that has a high antigenaffinity.
 17. A method for producing low affinity antibodies specificfor an antigen of interest comprising: a) selecting a B-cell capable ofproducing antibody specific for said antigen of interest or selecting aB-cell capable of differentiating into a B-cell which is capable ofproducing antibody specific for said antigen of interest; b) inducing,enhancing and/or maintaining expression of BCL6 in said B-cell; c)inducing, enhancing and/or maintaining expression of an anti-apoptoticnucleic acid in said B-cell; d) allowing expansion of said B-cells intoa first population of B-cells; e) performing antigen staining and BCRstaining of B-cells from said first population; f) selecting a B-cellthat has a low antigen affinity; g) allowing expansion of said selectedB-cell into a second population of B-cells; h) measuring the antigenassociation rate and dissociation rate of antibodies produced by atleast one B-cell from said second population; and i) selecting anantibody with an affinity for said antigen of interest that is lowerthan the average affinity of said first population of B-cells for saidantigen of interest.
 18. The method according to claim 1, wherein saidantigen association rate and dissociation rate in step h) is measuredusing surface plasmon resonance (SPR).